Sex chromatin preparation test can take oral mucous membrane cells, shed cells in urine, amniotic fluid cells, chorionic villus cells, etc. as the material for examination, and can detect X chromatin and Y chromatin at the same time.
Principle
The basic principle of the sex chromatin preparation experiment is that in the nucleus of normal female interphase cells, there is a darker oval-shaped vesicle immediately adjacent to the inner edge of the nuclear membrane, i.e., X chromatin, which is formed by the inactivation of random Lyonization of one of the X chromosomes in female cells.
This inactivation ensures that there is only one active chromosome in both male and female cells, and keeps the number of X-linked gene products at the same level in both sexes, which is known as X-chromosome dosage compensation.
X chromatin is unique to normal female cells, where the number of X chromatin is one less than the number of X chromosomes, and Y chromatin is the heterochromatin region of the long arm of Y chromosome in normal males that forms strongly fluorescent vesicles when stained with fluorescent dyes during interphase.
Operation method
Sex chromatin preparation experiment
Principle
The basic principle of the sex chromatin preparation experiment is that in the nucleus of normal female interphase cells, there is a darker oval-shaped vesicle immediately adjacent to the inner edge of the nuclear membrane, i.e., X chromatin, which is formed by the random Lyonization inactivation of one of the X chromosomes in the female cells. This inactivation ensures that there is only one active chromosome in the cells of both sexes, so that both sexes have the same number of X-linked gene products, which is known as dosage compensation of X chromosome. X chromosome is unique to normal female cells, and the number of X chromosome is one less than the number of X chromosome in the cells. Y chromosome is a heterochromatin region on the long arm of the Y chromosome in normal males, and it forms a strong chromatin in the nucleus after staining with a fluorescent dye during interphase. Y chromatin is the heterochromatin region of the long arm of the normal male Y chromosome that is stained by fluorescent dyes during interphase to form strongly fluorescent vesicles.
Materials and Instruments
Equipment: Move The basic process of sex chromatin preparation experiment can be divided into the following steps: (1) Oral mucosa cells A Let the examinee rinse the mouth with 0.85% saline several times to remove bacteria and other debris in the mouth as far as possible. B The operator holds the lower lip of the subject with one hand, and scrapes the mucosa on both sides of the cheek or the inner side of the lower lip with a wooden (or metal) tongue depressor or the blunt end of a toothpick (discard the cells scraped in the first scraping). C The same area was scraped several times in succession, and the scrapings were swished into a centrifuge tube containing 5 mL of 0.85% saline. D Centrifuge at 1500 r/min for 10 min, discard the supernatant and leave the cell mass. E Add 10 mL of freshly prepared fixative (3 parts methanol: 1 part glacial acetic acid) and mix gently to make a suspension. Fix for 30 min. F Centrifuge at 1500 r/min for 10 min, discard the supernatant and leave the cell mass. G Add several drops of freshly prepared fixative according to the number of cell clusters and mix thoroughly to make a suspension. H Take a drop of the suspension onto a pre-cooled clean slide and let it dry. (2) Exfoliated cells in urine A Ask the subject to drain the urine into a clean beaker or bottle. B Stir the urine and aspirate 10 mL of urine into a centrifuge tube, centrifuge at 1500-2000 r/min for 10 min, discard the supernatant and leave the cell mass. C Add 10 mL of newly prepared fixative (3 parts methanol: 1 part glacial acetic acid) and fix for 30 min. D Centrifuge at 1500 r/min for 10 min, discard the supernatant and leave the cell mass. E According to the number of cells, add several drops of newly prepared fixative and mix thoroughly to make a suspension. F Take a drop of the suspension onto a pre-cooled clean slide and let it dry. (3) Amniotic fluid cells, which are cells from the fetus, can be extracted for X chromosome examination to make a prenatal diagnosis of the sex of the fetus. A. About 10 mL of amniotic fluid from a pregnant woman in the 16th week of pregnancy should be punctured through the wall according to gynecological practice (when extracting, 2-3 mL of amniotic fluid should be discarded to avoid contamination by maternal cells). B Centrifuge at 1500 r/min for 10 min and discard the supernatant. C Add 10 mL of newly prepared fixative (3 parts methanol: 1 part glacial acetic acid) to the cell mass, mix well to form a cell suspension, and fix for 30 min at room temperature. D Centrifuge at 1500 r/min for 10 min and discard the supernatant. E Add a few drops of freshly prepared fixative according to the number of cells and mix well to make a suspension. F Take a drop of the suspension onto a pre-cooled clean slide and let it dry. (1) Corynebacterium sulphide staining A Place the slide specimen in 1 mol/L HCl and hydrolyze for 20 min at 37 ℃. B Rinse well with distilled water and air dry. C Fixed slide specimens were immersed in Corydalis staining solution for about 15 min. D Rinse with distilled water and air dry. E Look for concentrated and evenly dispersed cells under low magnification, and then turn to oil microscope for observation. (2) Crystal violet staining method A Fixed slide specimens were treated with 70% and 50% ethanol (with two changes of distilled water) for 5 minutes each time. B. Immerse the specimen in 1% crystal violet solution for 5-8 min (0.5% crystal violet solution can be used for mass production, and stain for 10-15 min). C. Differentiate the slide specimen in 95% ethanol (rapidly perform 5-8 baptisms in 95% ethanol). D Differentiate the slide specimen in anhydrous ethanol, examining it intermittently under a microscope until the microscopic parts of the nuclear structure are clear (usually about 1 min). E Immerse in xylene. Replace the xylene twice for 3 min each to make the specimen transparent. F Resin seal. G Look for concentrated and evenly dispersed cell clusters under low magnification before turning to oil microscope for observation. (3) Carbonic acid-fuchsin staining method A Drops of Carbonic Acid-Fuchsin working solution onto the slide specimen and stain for 5 minutes. B Dehydrate with 95% and 100% ethanol for 30 s each. C. Look for concentrated and evenly dispersed cells under low magnification, and then turn to oil microscope for observation. A Prepare 0.005% azadirachtin quinacrine solution in pH 6.0 Mcllvaine buffer (ready to use), and put it into a dark color bottle (can be stored in the refrigerator at 4 ℃ for two weeks). B Immerse the slide specimen in pH 6.0 Mcllvaine buffer for a few seconds. C Immerse the slide specimen in 0.005% azathioprine quinacrine solution for more than 10 min. D Place the slide specimen in pH 6.0 Mcllvaine buffer (or distilled water) for 10 min for color separation. Caveat 1 When scraping the cells from the oral mucosa, the tongue depressor or toothpick should be scraped in the same direction, not back and forth, so as not to damage the oral mucosa.2 Urine can be prevented at room temperature for 6 h without changing the cell morphology. However, it is better to make the specimen immediately after taking the material to avoid contamination of bacteria or deterioration of the cells due to the long period of time.3 When observing Y chromatin, it should be noted that female cells may also show bright fluorescent vesicles, but the size and brightness of the fluorescent dots of these cells are not consistent, so they should be distinguished. For more product details, please visit Aladdin Scientific website.
① Slide
② Staining vat
③ Tongue depressor (or sterilized toothpicks)
④ Microscope
⑤ Fluorescence microscope
⑥ Human oral mucosa cells
⑦ Urine
⑧ Amniotic fluid.
Reagents:
① 0.85% saline
② fixed solution (3 methanol: 1 glacial acetic acid, ready to use)
③ 1 mol/L HCl
③ 1 mol/L HCl ④ Corydalis staining solution
⑤ 1% Crystalline violet dye
⑥ Carbonic acid-red dye
⑦ Anhydrous ethanol
⑧ 95%/70%/50% gradient ethanol
⑨ Xylene
⑩ Mcllvaine buffer
⑪ Nitrogen mustard quinacrine (QM)